The catalytic active site of the Polymerization Domain (PolDom) of bacterial Ligase D is designed to promote realignments of the primer and template strands and extend mispaired 3' ends. These features, together with the preferred use of ribonucleotides (NTPs) over deoxynucleotides (dNTPs), allow PolDom to perform efficient double strand break repair by nonhomologous end joining when only a copy of the chromosome is present and the intracellular pool of dNTPs is depleted. Here, we evaluate (i) the role of conserved histidine and serine/threonine residues in NTP insertion, and (ii) the importance in the polymerization reaction of a conserved lysine residue that interacts with the templating nucleotide. To that extent, we have analyzed the biochemical properties of variants at the corresponding His651, Ser768, and Lys606 of Pseudomonas aeruginosa PolDom (Pa-PolDom). The results show that preferential insertion of NMPs is principally due to the histidine that also contributes to the plasticity of the active site to misinsert nucleotides. Additionally, Pa-PolDom Lys606 stabilizes primer dislocations. Finally, we show that the active site of PolDom allows the efficient use of 7,8-dihydro-8-oxo-riboguanosine triphosphate (8oxoGTP) as substrate, a major nucleotide lesion that results from oxidative stress, inserting with the same efficiency both the anti and syn conformations of 8oxoGMP.
Keywords: DNA repair; archaea/eukaryotic primase; ligase D; nonhomologous end joining (NHEJ).